US20230241155A1 - Methods and means for modifying hemodynamics in infections - Google Patents

Methods and means for modifying hemodynamics in infections Download PDF

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US20230241155A1
US20230241155A1 US17/995,692 US202117995692A US2023241155A1 US 20230241155 A1 US20230241155 A1 US 20230241155A1 US 202117995692 A US202117995692 A US 202117995692A US 2023241155 A1 US2023241155 A1 US 2023241155A1
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peptide
aqgv
infection
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Gert Wensvoort
Eric Claassen
Johan Renes
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Biotempt BV
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

  • the application relates to methods and means for alleviating certain effects resulting from infection, in particular, hemodynamic effects. More specifically, the disclosure relates to peptide preparations used in the treatment of viral infections that affect the permeability of the vascular system.
  • SARS-Cov-2 a coronavirus causing COVID-19.
  • the new coronavirus mainly seems to kill by flooding and clogging the tiny air sacs in the lungs with fluid, choking off the body's oxygen supply until it shuts down the organs essential for life.
  • Such suffocation with one's own fluid seems a model of respiratory disease that more coronaviruses may be capable of inducing.
  • a large reservoir of such viruses in various exotic animals may cause a similar pandemic with similar suffocation as SARS-Cov-2, considering our lack of pre-existing immunity.
  • the disclosure provides a method for reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, such as with a virus.
  • the disclosure provides the method for reducing the gas diffusion distance between lung-alveoli and the vascular network surrounding alveoli in a human subject suffering from a respiratory infection, allowing reduction of fluid in alveoli and/or allowing improved oxygen supply to the subject's body.
  • the substance comprises an AQGV-peptide (SEQ ID NO:2), an LQGV-peptide(SEQ ID NO:3), or a functional analogue of either.
  • the disclosure provides the method for reducing the gas diffusion distance between lung-alveoli and the vascular network surrounding alveoli in a human subject suffering from an infection with a respiratory virus. It is moreover preferred that the viral infection is caused by a virus requiring a specific receptor and a more ubiquitous binding partner present on at least a percentage of lung alveolar cells. In one preferred embodiment, it is preferred that the specific receptor is ACE-2. In another preferred embodiment, it is preferred that the more ubiquitous binding partner is a glycoprotein comprising a sialic acid residue.
  • the ubiquitous binding partner binds with a fMLF-like amino acid sequence, for example, wherein the sequence at least comprises a membrane-proximal-external-region (MPER, herein also identified as fusogenic sequence(SEQ ID NO:1)).
  • MPER membrane-proximal-external-region
  • the virus is a coronavirus, in particular, a coronavirus with an MPER as identified in FIG. 11 , more in particular, at least comprising a fusogenic sequence as identified in FIG. 12 .
  • the 1VIPER at least comprises amino acid sequence KWPWIWL (amino acids identified herein by one-letter code) (SEQ ID NO:1).
  • the disclosure provides a method for reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, such as with a virus, wherein the coronavirus is the COVID-19 virus (SARS-COV-2) or a mutant thereof.
  • SARS-COV-2 COVID-19 virus
  • the disclosure provides a method for reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, such as with a virus.
  • the disclosure provides the method for reducing the gas diffusion distance between lung-alveoli and the vascular network surrounding alveoli in a human subject suffering from a respiratory infection, allowing reduction of fluid in alveoli and/or allowing improved oxygen supply to the subject's body.
  • the substance comprises an AQGV-peptide(SEQ ID NO:2), an LQGV-peptide(SEQ ID NO:3), or a functional analogue of either.
  • the disclosure provides the method for reducing the gas diffusion distance between lung-alveoli and the vascular network surrounding alveoli in a human subject suffering from an infection with a respiratory virus. It is moreover preferred that the viral infection is caused by a virus requiring a specific receptor and a more ubiquitous binding partner present on at least a percentage of lung alveolar cells. In one preferred embodiment, it is preferred that the specific receptor is ACE-2. In another preferred embodiment, it is preferred that the more ubiquitous binding partner is a glycoprotein comprising a sialic acid residue.
  • the ubiquitous binding partner binds with a fMLF-like amino acid sequence, for example, wherein the sequence at least comprises a membrane-proximal-external-region (MPER, herein also identified as fusogenic sequence).
  • MPER membrane-proximal-external-region
  • the virus is a coronavirus, in particular, a coronavirus with an MPER as identified in FIG. 11 , more in particular, at least comprising a fusogenic sequence as identified in FIG. 12 .
  • the MPER at least comprises amino acid sequence KWPWIWL ((SEQ ID NO:1)amino acids identified herein by one-letter code).
  • the disclosure provides a method for reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, such as with a virus, wherein the coronavirus is the COVID-19 virus (SARS-COV-2) or a mutant thereof.
  • SARS-COV-2 COVID-19 virus
  • the disclosure also provides a method for reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, wherein the substance is administered intravenously to the subject, preferably at a rate of at least 75 mg/kg/hour, or more preferably at least 90 mg/kg/hour. It is moreover preferred that the substance is administered intermittently. It is moreover preferred that during treatment the subject is monitored for hemodynamic stability.
  • the disclosure also provides a method for reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, wherein the substance is administered intravenously to the subject, the method further comprising administering an antiviral agent, such as remdesivir (GS-5734), an inhibitor of the viral RNA-dependent, RNA polymerase, to the subject.
  • an antiviral agent such as remdesivir (GS-5734)
  • the disclosure also provides a method for reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, wherein the substance is administered intravenously to the subject, the method further comprising administering an anti-inflammatory agent, such as dexamethasone or an interleukin-6 signaling inhibitors such as tocilizumab, to the subject.
  • an anti-inflammatory agent such as dexamethasone or an interleukin-6 signaling inhibitors such as tocilizumab
  • the disclosure also provides a pharmaceutical formulation for use in a method for reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, wherein the substance is administered intravenously to the subject, preferably at a rate of at least 75 mg/kg/hour, or more preferably at least 90 mg/kg/hour. It is moreover preferred that the substance is administered intermittently. It is moreover preferred that during treatment the subject is monitored for hemodynamic stability.
  • the disclosure also provides a pharmaceutical formulation for use in reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, wherein the substance is administered intravenously to the subject, the method further comprising administering an antiviral agent, such as remdesivir (GS-5734), an inhibitor of the viral RNA-dependent, RNA polymerase, to the subject.
  • an antiviral agent such as remdesivir (GS-5734)
  • the disclosure also provides a pharmaceutical formulation for use in reducing the permeability of an endothelial layer of a blood vessel in a subject, the method comprising providing to the endothelial layer a substance that reduces the ratio of Angiopoietin-2 to Angiopoietin-1 at the site of increased permeability as a result of an infection, wherein the substance is administered intravenously to the subject, the method further comprising administering an anti-inflammatory agent, such as dexamethasone or an interleukin-6 signaling inhibitors such as tocilizumab, to the subject.
  • an anti-inflammatory agent such as dexamethasone or an interleukin-6 signaling inhibitors such as tocilizumab
  • the disclosure also provides a pharmaceutical formulation for use according to the invention, comprising an AQGV-peptide(SEQ ID NO:2), an LQGV-peptide(SEQ ID NO:3), or a functional analogue of either and an excipient suitable for parenteral administration.
  • a human subject When a human subject is suffering from an infection, in particular, a viral infection, more in particular, a viral respiratory infection, many effects are seen that influence hemodynamic stability.
  • One of the effects seen is increased permeability of blood vessels leading to fluid leakage from blood vessels into the intercellular spaces, and vice versa, resulting in aggravated and traumatic damage to the lung, and other organs.
  • Typical signs of such suffocating damage induced by leakage from vessels include increased extra-cellular fluid in lungs with fluid overflow into alveoli.
  • thrombosis may be seen, in particular, leading to (deep) venous thrombosis ((D)VT) and pulmonary embolism (PE).
  • the alveolar-capillary membrane This is the thin layer between the small air sacks in the lung (the alveoli) and the smallest blood vessels that travel through the lungs (lung-capillaries). How well oxygen is inhaled and can pass (diffuse) from the alveoli into the blood, and how well carbon dioxide can pass from the blood capillaries back into the alveoli to be exhaled, depends on how thick (swollen) this membrane is, and how much surface area is available for the transfer to take place.
  • Diffusing capacity may be low if there is less surface area available for the transfer of oxygen and carbon dioxide, for example, with emphysema, or if a lung or part of it is removed for lung cancer, or PE and or pre-existing cardiovascular and metabolic issues and obesity.
  • diffusing capacity may be low if lung disease is present that causes the membrane to be thicker, for example, in chronic lung disease such as pulmonary fibrosis, as, for example, seen with COPD, and with sarcoidosis.
  • the present disclosure is particularly useful for such patients having only partial lung capacity.
  • Acute disease can also result in low diffusing capacity, for example, in aggravated viral respiratory infections with injury to the lung, often the permeability of lung-capillaries is increased generating a flux of fluid from the capillaries into the thin layer of extra-cellular-matrix separating alveoli from capillaries, with intercellular fluid retention therewith thickening (swelling) the membrane through accumulation of fluid in the extra-cellular-matrix (interstitium) separating alveolar cells from vascular cells.
  • plasma levels of biomarkers of endothelial activation are often predictive of mortality and morbidity.
  • angiopoietin-2 may be a useful biologic marker of mortality in acute lung injury (ALI) patients.
  • D-dimer plasma levels may be used to follow a patient's health status in aggravated viral respiratory infections with injury to the lung and endothelial activation.
  • D-dimer the lysis product of cross-linked fibrin indicates fibrinolysis in response to clotting activation and fibrin formation (doi.org/10.1111/jth.12075).
  • D-dimer levels are evident in febrile and convalescent phases typically following viral infections that affect vascular endothelial cells and associate with endothelial activation and plasma leakage.
  • D-dimer assays can vary in sensitivity depending on the lab-specific type drawn, and not all labs report the same units providing various acceptable ranges for the results.
  • VTE venous thromboembolism
  • the disclosure in one aspect provides a method for reducing the permeability of an endothelial layer of a blood vessel comprising providing to the layer a substance reducing the ratio of angiopoietin-2 to angiopoietin-1 at the site of increased permeability as a result of an infection.
  • this method serves to reduce the gas diffusion distance (or at least to prevent increasing the diffusion distance) between lung-alveoli and the vascular network surrounding alveoli in a human subject suffering from a respiratory infection, in particular, patients with underlying disease causing limited oxygen availability.
  • Reduced vascular permeability in patients suffering vascular leakage is generally associated with reduced D-dimer levels.
  • the substances to be used in the methods according to the disclosure include peptides that influence hemodynamics, particularly by influencing gap junctions between the cells.
  • Such peptides include AQGV (SEQ ID NO:2) and functional analogues thereof.
  • a functional analogue is defined as a substance that provides the same or a similar function (in kind, not necessarily in amount).
  • any substance that decreases permeability of the vascular system may be used according to the present invention.
  • tetrapeptide AQGV ((SEQ ID NO:2) herein also referred to as EA-230) has surprisingly been found to modulate vascular permeability to the good.
  • EA-230 can be used to reduce the infection-associated occurrence of adverse fluid in the lungs, reduce hypoxemia, reduce PE, and therewith also reduce ventilator use with its detrimental systemic effects, in particular, in viral respiratory infections such as caused by influenza viruses and in particularly by coronaviruses.
  • the active substance to control hemodynamic stability comprises an AQGV peptide(SEQ ID NO:2).
  • a functional and/or structural AQGV analogue according to the disclosure may be selected from the group consisting of peptides comprising a tetrapeptide selected from the group of AQLP (SEQ ID NO:4), PLQA (SEQ ID NO:5), LQGV (SEQ ID NO:3), LAGV (SEQ ID NO:7), PQVG (SEQ ID NO:8), PQVA (SEQ ID NO:9), PQVR (SEQ ID NO:10), VGQL (SEQ ID NO:11), LQPL (SEQ ID NO:12), RQGV (SEQ ID NO:13), LQVG (SEQ ID NO:14), LQGA (SEQ ID NO:15), LQGR (SEQ ID NO:16), AQGA (SEQ ID NO:17), QPLA (SEQ ID NO:18), PQVP (SEQ ID NO:19), VGQA (SEQ ID NO:20), QVGQ (SEQ ID NO:21), VGQG (SEQ ID NO:22)
  • the disclosure further provides a method wherein the viral infection is caused by a virus requiring a specific receptor and a more ubiquitous binding partner present on at least a percentage of lung alveolar cells.
  • the viral infection is caused by a coronavirus wherein the specific receptor is ACE-2, in particular, wherein the coronavirus is SARS-Cov-2 or a mutant or analogue thereof.
  • Other coronavirus infections that may be treated according to the disclosure carry specific receptor DPP4 (such as with MERS corona virus) or APN (aminopeptidase N).
  • the more ubiquitous binding partner is a glycoprotein comprising a sialic acid residue.
  • the ubiquitous binding partner binds with a fMLF-like amino acid sequence, for example, wherein the sequence at least comprises a membrane-proximal-external-region (MPER, herein also identified as fusogenic sequence).
  • MPER membrane-proximal-external-region
  • the virus is a coronavirus.
  • the virus is a coronavirus, in particular, a coronavirus with an MPER as identified in FIG. 11 , more in particular, at least comprising a fusogenic sequence as identified in FIG. 12 .
  • the more ubiquitous binding partner is a glycoprotein comprising a sialic acid residue as recognized by influenza virus.
  • AQGV peptide or related substance is administered intravenously, preferably at a rate of at least 75 mg/kg/hour, more preferably at least 90 mg/kg/hour. It is, in particular, useful to administer the AQGV peptide or related substance intermittently.
  • Preferred use is dosing for 2-4 hours at at least 90 mg/kg/hour, then reducing to 30 mg/kg/hour for 2-4 hours, or for as long as it takes to monitor the patients response to treatment by clinical or laboratory diagnosis, or stop administering the substance for 1-2 hours until diagnostic studies such as point-of-care testing have completed, and then resume treatment with 2-4 hours at at least 90 mg/kg/hour. It is preferred that the monitoring comprises studying the subject for hemodynamic stability and/or fibrinolysis. Treatment with AQGV peptide (SEQ ID NO:2) according to the disclosure may further comprise administering an antiviral agent.
  • the disclosure also provides a pharmaceutical formulation comprising an AQGV peptide (SEQ ID NO:2) or related substance (preferably a functional analogue) for use in a method according to the invention, or a pharmaceutical formulation for use according to the invention, comprising an AQGV peptide (SEQ ID NO:2), or a functional analogue thereof and an excipient suitable for parenteral administration.
  • an AQGV peptide SEQ ID NO:2
  • related substance preferably a functional analogue
  • the human subject or patient experiencing reduced diffusion may be admitted into an intensive care unit (ICU) where vital signs are monitored.
  • ICU intensive care unit
  • the patient receives medical treatment to allow the patient to recover and when vital signs are within acceptable boundaries, the patient can be released from ICU and admitted into standard hospital care.
  • the patient can be released from the hospital and returns home.
  • a patient can be readmitted into the hospital should the need arise because, e.g., the condition or infection status of the patient worsens.
  • Any improvement on the health and recovery of a patient affecting the length of stay of a patient in the ICU, the length of stay in standard care at the hospital and/or patient re-admittance provides for a significant benefit to patients.
  • any means and methods according to the disclosure that improve the health and, in particular, speed of recovery of a patient through use of the peptide compounds disclosed herein are of interest.
  • an AQGV (SEQ ID NO:2) peptide also referred to as EA-230 herein
  • the peptide was found to be safe but, unexpectedly, no immunomodulatory effects were observed under the test circumstances when comparing treated patients as compared with control subjects.
  • the current inventors surprisingly found that upon analysis of the data obtained in the clinical trial, new and highly advantageous properties could be attributed to the AQGV (SEQ ID NO:2) peptide, which have not been observed before. These properties are apparently independent from known and observed immunomodulatory effects.
  • the current disclosure relates to the use of an AQGV (SEQ ID NO:2) peptide, and analogues thereof, for improving the clinical parameters of human patients admitted into hospital and/or intensive care such that the time period between admittance and release from hospital and/or intensive care can be shortened.
  • the use of an AQGV (SEQ ID NO:2) peptide, and analogues thereof is for use in a medical treatment for modifying hemodynamics in human subjects.
  • the use in human subjects for modifying hemodynamics involves a reduction of reducing undesired fluid retention and/or a reduced use of vasopressive agents in the human subject.
  • the use of an AQGV (SEQ ID NO:2) peptide, and analogues thereof is for use in human subjects having impaired lung function.
  • an AQGV SEQ ID NO:2
  • a functional analogue thereof is provided for use in a method of treatment of a human subject, wherein the use comprises a treatment for modifying hemodynamics in the human subject.
  • Hemodynamics involves the dynamics of blood flow, i.e., the physical factors that govern blood flow through the human body. Hemodynamics in human patients can be monitored by measuring, e.g., blood pressure and/or the fluid balance. When blood pressure is low and/or the fluid balance disturbed in a human patient, vasopressors, or inotropes may be used and/or fluid administered, e.g., intravenously.
  • Inotropes and vasopressors are biologically and clinically important vasoactive medications that originate from different pharmacological groups and act at some of the most fundamental receptor and signal transduction systems in the body. More than 20 such agents are in common clinical use, yet few reviews of their pharmacology exist outside of physiology and pharmacology textbooks. Despite widespread use in critically ill patients, understanding of the clinical effects of these drugs in pathological states is poor. Adverse effects of vasopressors and inotropes depend on the mechanism of action. For the medications that have beta stimulation, arrhythmias are one of the most common adverse effects that one would like to reduce.
  • the current inventors have found that by using an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, the hemodynamics in human patients post-trauma (e.g., viral infection) was significantly improved as shown by, e.g., a reduced use of vasopressors and/or an improved fluid balance in human patients.
  • the use of an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, as described herein thus improves the hemodynamic stability in human patients. Modifying or optimizing hemodynamics in human subjects is of importance post-injury, when, e.g., human subjects have suffered infection, trauma and/or blood loss.
  • the AQGV (SEQ ID NO:2) peptide, or an analogue thereof can advantageously be used in hemodynamic therapy.
  • Hemodynamic therapy comprises the optimization of hemodynamics in patients in goal-directed hemodynamic therapy.
  • Such therapies can include therapeutic interventions such as fluid management in patients and/or the use of vasopressors.
  • AQGV (SEQ ID NO:2) functional analogues are defined herein as peptides exerting analogous effect or function as the AQGV (SEQ ID NO:2) peptide as described herein, in kind not necessarily in amount.
  • the AQGV (SEQ ID NO:2) peptide has a length of 4 amino acids.
  • An AQGV (SEQ ID NO:2) functional analogue may have sequence identity, i.e., comprising at least part or the whole of the AQGV (SEQ ID NO:2) peptide.
  • such an AQGV (SEQ ID NO:2) functional analogue is a structural analogue of the AQGV (SEQ ID NO:2) peptide.
  • a preferred structural analogue may be an LQGV (SEQ ID NO:3) peptide.
  • Structural analogues of the AQGV (SEQ ID NO:2) peptide may be selected from peptides comprising amino acids selected from the group of amino acids alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), proline (P) and arginine (R).
  • a AQGV (SEQ ID NO:2) structural analogue that comprises at least 50%, more preferably at least 75%, most preferably at least 100% amino acids selected from the group of autophagy-inhibiting amino acids alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), proline (P), and arginine (R).
  • a structural analogue of the AQGV (SEQ ID NO:2) peptide has a length in the range of 4-12 amino acids.
  • such a structural analogue is a linear peptide.
  • Suitable structural analogues of AQGV may have a length less than 4, e.g., of 3, however such lengths may require higher doses of such peptides because the half-life of such peptides will be shorter and thus less preferred. Longer structural analogues, e.g., longer than 12 residues, are less preferred because of potential immunogenicity of such longer peptides.
  • a structural AQGV (SEQ ID NO:2) analogue according to the disclosure may be selected from the group consisting of peptides comprising a tetrapeptide selected from the group of AQLP (SEQ ID NO:4), PLQA (SEQ ID NO:5), LQGV (SEQ ID NO:6), LAGV (SEQ ID NO:7), PQVG (SEQ ID NO:8), PQVA (SEQ ID NO:9), PQVR (SEQ ID NO:10), VGQL (SEQ ID NO:11), LQPL (SEQ ID NO:12), RQGV (SEQ ID NO:13), LQVG (SEQ ID NO:14), LQGA (SEQ ID NO:15), LQGR (SEQ ID NO:16), AQGA (SEQ ID NO:17), QPLA (SEQ ID NO:18), PQVP (SEQ ID NO:19), VGQA (SEQ ID NO:20), QVGQ (SEQ ID NO:21), and VGQG (SEQ ID
  • Vasopressors are a class of drugs that can elevate low blood pressure. Some vasopressors act as vasoconstrictors, other vasopressor sensitize adrenoreceptors to catecholamines-glucocorticoids, and another class of vasopressors can increase cardiac output. Whichever vasopressor is used, the current disclosure allows for a reduction in the use of vasopressors. A reduction in the use of vasopressors involves a reduction of the duration of vasopressor use and/or a reduction of the dosage of the vasopressor.
  • vasopressors are, e.g., epinephrine, noradrenaline, phenylephrine, dobutamine, dopamine, and vasopressin.
  • Fluid management in patients involves monitoring, e.g., oral, enteral, and/or intravenous intake of fluids and fluid output (e.g., urine) and subsequently managing fluid intake, e.g., in case of an observed fluid retention (i.e., the fluid intake exceeds fluid output).
  • fluids and fluid output e.g., urine
  • fluid output e.g., urine
  • fluid intake e.g., urine
  • AQGV SEQ ID NO:2
  • the AQGV (SEQ ID NO:2) peptide, or analogue thereof can be used in addition to known interventions that are to improve the hemodynamics in human patients, thereby resulting in a faster improvement in hemodynamics as compared with not using an AQGV (SEQ ID NO:2) peptide, or an analogue thereof.
  • an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof is provided for use in the treatment of a human subject having impaired lung function.
  • the impaired lung function is acute lung injury.
  • an AQGV peptide, or a functional analogue thereof is provided for use in the treatment of a human subject for improving lung function.
  • Lung function can also be assessed by measuring hypoxemia, or by determining the alveolar-arterial gradient (A-aO2, or A-a gradient). Assessing A-a gradient to assess lung function in humans is standard clinical practice (e.g., by determining the difference between the alveolar concentration (A) of oxygen and the arterial (a) concentration of oxygen.
  • the A-a gradient helps to assess the integrity of the alveolar capillary unit. Improvements in lung function as compared with not receiving the AQGV (SEQ ID NO:2) peptide can include progressing to a lung function stage to a less severe stage (e.g., a patient progressing from having lung injury to being at risk of lung injury or having no lung injury). Irrespective of what assessment is made, the use of the AQGV (SEQ ID NO:2) peptide, or analogue thereof, can improve lung function in humans having lung injury and/or an impairment of lung function in subjects absent of immunomodulatory effects.
  • the use of the AQGV (SEQ ID NO:2) peptide allows for improving lung function, however, it can also prevent a reduction and/or an impairment of lung function. Accordingly, lung injury with hypoxemia may be prevented.
  • the use of the AQGV (SEQ ID NO:2) peptide, or analogue thereof allows to maintain lung function in human patients.
  • the use of the AGQV peptide, or analogue thereof allows to protect lung function in human patients.
  • the use of the AQGV (SEQ ID NO:2) peptide, or analogue thereof allows to prevent a reduction and/or impairment of lung function in human patients.
  • a human patient that can be classified as having no lung injury, or being at risk of having lung injury (such as with COVID-19), may receive treatment with the AQGV (SEQ ID NO:2) peptide, whereby such a patient may maintain its status instead of progressing to a (more severely) reduced lung function.
  • human patients that are at risk of developing lung injury e.g., due to (induced) trauma, such as infection, may, as a result of receiving treatment with the AQGV (SEQ ID NO:2) peptide, or analogue thereof, maintain their lung function status.
  • an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof is provided for use in the treatment of a human subject having impaired lung function whereby the use comprises modifying hemodynamics in the human subject.
  • an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, in accordance with the disclosure can advantageously be used to protect lung function and/or improve lung function, and modifying hemodynamics.
  • Such combined use resulting, e.g., in improved and/or maintained lung function and a reduction in the use of vasopressors and/or improved fluid management in human subjects.
  • the current disclosure provides for a reduced use of vasopressive agents.
  • the use of vasopressive agents can be reduced by reducing the duration of the use of vasopressive agents.
  • the use of vasopressive agents can be reduced by reducing the amount of vasopressive agents (e.g., reducing amount per dosage and/or increasing time interval between administrations).
  • the use of vasopressive agents can be reduced by reducing the amount of vasopressive agents and the duration of the use of vasopressive agents.
  • an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof reduces adverse fluid retention in the human subject.
  • Fluid retention can occur in human subjects, symptoms of which, e.g., include weight gain and edema. Fluid retention can be the result of reduced lung function and/or impaired hemodynamics.
  • the use of AQGV can affect lung function and/or hemodynamic stability in human subjects, the use of AQGV (SEQ ID NO:2) can affect fluid retention as well. Fluid retention can be the result of leaky capillaries.
  • AQGV AQGV
  • analogues thereof may have an effect on the leakiness (permeability) of capillaries, reducing leakage of plasma from the blood to peripheral tissue and/or organs.
  • Most preferably edema can be reduced and/or avoided by the use of AQGV (SEQ ID NO:2).
  • Such may also be referred to as adverse fluid retention as it has an adverse effect on the patient.
  • the use of an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof can improve fluid retention dynamics in human subjects thereby alleviating symptoms associated with fluid retention such as weight gain and edema, which subsequently can reduce the use of diuretics.
  • the use of the AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, in accordance with the invention is not restricted to patients having lung injury and/or requiring hemodynamic therapy.
  • the use of an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, in accordance with the invention includes the treatment of human patients that are believed to be at risk of having lung injury and/or anticipated to require hemodynamic therapy. Such human patients include patients that are to be admitted, or are expected to be admitted, into intensive care.
  • the use of the AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof includes a use for trauma, such as infection, as exemplified, e.g., in the examples.
  • the use of the AQGV (SEQ ID NO:2) peptide for trauma, such as infection may be before, but is typically during and/or after infection. It may be preferred that the use of the AQGV (SEQ ID NO:2) peptide, or an analogue thereof, is during infection with a virus.
  • the use of AQGV (SEQ ID NO:2) peptide as is provided herein is, in particular, useful in patients subjected to a long duration of mechanical ventilation, i.e., longer than 2.5 hours.
  • the use of the AQGV (SEQ ID NO:2) peptide, or an analogue thereof is during a mechanical ventilation of longer than 2.5 hours and wherein the AQGV (SEQ ID NO:2) peptide or analogue thereof is administered during the mechanical ventilation.
  • the use of an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, for use in accordance with the disclosure is for use is in a human subject having COVID-19. It is well known that shortening the duration of mechanical ventilation is highly correlated with recovery and prevention of re-admittance of patients.
  • the use of the AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, in accordance with the disclosure and as described above, involves the administration of the peptide into the bloodstream.
  • administration into the bloodstream comprises, e.g., intravenous administration or intra-arterial administration.
  • a constant supply of AQGV (SEQ ID NO:2) peptide, or an analogue thereof, is preferred, e.g., via an infusion wherein the AQGV (SEQ ID NO:2) peptide, or analogue thereof, is comprised in a physiological acceptable solution.
  • Suitable physiological acceptable solutions may comprise physiological salt solutions (e.g., 0.9% NaCl) or any other suitable solution for injection and/or infusion.
  • physiological solutions may comprise further compounds (e.g., glucose etc.) that may further benefit the human subject, and may also include other pharmaceutical compounds (e.g., vasopressors).
  • the AQGV (SEQ ID NO:2) peptide is administered at a rate that is at least 50 mg/kg patient weight per hour (mg/kg/hr).
  • the administration rate is at least 60 mg, at least 70, at least 80 or, most preferably, at least 90 mg/kg/hr.
  • the AQGV (SEQ ID NO:2) peptide is administered for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours.
  • the administration of the AQGV (SEQ ID NO:2) peptide is at a rate of at least 70 mg/kg/hr. and administered for at least 1 hour, more preferably at least 1.5 hours, most preferably at least 2 hours.
  • the administration is during infection. More preferably, the administration is during essentially the entire duration of disease resulting from infection.
  • treatment will start after determination of a level of severity justifying the treatment.
  • the treatment may typically last from its detection until the absence of detectable infection or sufficient recovery to allow for end of treatment.
  • the mean arterial maximum concentrations (mean Cmax) as determined in vivo in humans for EA-230 in the Phase II clinical trial was 30500 ng/mL, in the range of 12500 to 57500 ng/mL.
  • the mean venous Cmax found was 68400 ng/mL, in the range of 19600 to 113000 ng/mL.
  • whichever means and methods are used for administration of EA-230 (or AQGV) preferably, means and methods that allow obtaining an arterial Cmax in the range of 10,000 to 60,000 ng/mL and/or a venous Cmax in the range of 15000 to 120000 ng/ml can be contemplated.
  • the route of administration may not be necessarily be restricted to intravenous administration, but may include other routes of administration resulting in similar venous and/or arterial Cmax concentrations.
  • an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof is provided for any use in accordance with the disclosure as described above, wherein the human subject is admitted to intensive care, and wherein the use improves parameters measured of the human subject, the parameters of the human subject typically those being determined to assess whether the patient needs to remain in intensive care or not.
  • parameters that are assessed when a human patient is in intensive care include parameters related to lung function and hemodynamics.
  • the use of the AQGV (SEQ ID NO:2) peptide, or analogue thereof is to improve such parameters to thereby reduce the length of stay in the intensive care unit.
  • the effect of the use of the AQGV (SEQ ID NO:2) peptide, or analogue thereof also reduces the length of stay in the hospital and reduces re-admittance into the hospital.
  • the use of the AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof has a profound effect on lung function and/or hemodynamics in human subjects thereby advantageously benefiting human subjects when, e.g., suffering from induced trauma, e.g., when undergoing mechanical ventilation.
  • the use of the AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof is for use in patients subjected to mechanical ventilation.
  • the use of the AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof is for use in human patients experiencing or thought to be experiencing COVID-19 or a similar infection.
  • the disclosure relates to a distinct and new class of drugs: autophagy-inhibiting compounds that comprise peptides and/or amino acids that target the nutrient sensing system of the mechanistic target of rapamycine, mTOR and inhibit autophagy.
  • autophagy-inhibiting compounds that comprise peptides and/or amino acids that target the nutrient sensing system of the mechanistic target of rapamycine, mTOR and inhibit autophagy.
  • AQGV SEQ ID NO:2
  • an autophagy-inhibiting peptide herein also referred to as an AQGV (SEQ ID NO:2) peptide, and analogues (functional equivalents) thereof, for improving vascular permeability.
  • the effect of the AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof may have an effect on vasoconstriction.
  • Vasoconstriction involves the narrowing of the blood vessels resulting from contraction of the muscular wall of the vessel.
  • the use of an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, in accordance with the invention involves inducing vasoconstriction.
  • the disclosure also provides a method for identifying a peptide capable of reducing p38 MAPK kinase activity, comprising providing cells with a peptide comprising amino acids, the amino acids for at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably 100%, amino acids selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R), providing the cells with fMLP and detecting phosphorylation of p38 MAPK in the absence and presence of the peptide at an appropriate time interval, preferably in the order of minutes, most preferably from about half a minute to about 5 minutes, e.g., 30 to 600 seconds after provision of fMLP, and comparing the results to determine the peptide's effect on the phosphorylation.
  • alanine
  • FPR-activation of FPR-expressing cells with prototype FPR-ligand fMLP was detected to cause rapidly induced and significant (p ⁇ 0.05; p38 from 60 to 600 sec, PKB at 600 sec) changes in phosphorylation status of PKB (also known as AKT) ( FIG. 10 a ) and p38 MAPK kinases ( FIG. 10 c ), but not (or not detected) in STAT3, JNK ( FIG. 10 b ) and P42/p44MAPK/ERK1,2 ( FIG. 10 d ) kinases.
  • the disclosure also provides a method for identifying a peptide capable of reducing PI3K/AKT/mTOR activity, comprising providing cells with a peptide consisting of amino acids, the amino acids for at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably 100%, amino acids selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R), providing the cells with fMLP and detecting phosphorylation of PKB (AKT) in the absence and presence of the peptide at an appropriate time interval, preferably in the order of minutes, most preferably from about half a minute to about 5 minutes, e.g., 30 to 600.
  • alanine in one letter code: A
  • glutamine (Q) glycine
  • V valine
  • V leu
  • AQGV (SEQ ID NO:2) peptide effects on p38 MAPK ( FIG. 10 c ) are already detected at 30 seconds after FPR-stimulation, AQGV (SEQ ID NO:2) peptide effects on PKB(AKT) follow ( FIG. 10 a ) in a bi-phasic pattern at 300 sec. Both AQGV peptide effects on p38 and PKB-mediated signaling last for the full 600 seconds tested whereas the other kinases tested were not affected throughout.
  • AQGV SEQ ID NO:2
  • PI3K/AKT/mTOR activated induced changes in cell cytoskeleton reorganization affecting endothelial cell contraction and adverse vascular permeability.
  • the disclosure also provides a method for identifying a peptide capable of reducing PI3K/AKT/mTOR activity, comprising providing cells with a peptide consisting of amino acids, the amino acids for at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably 100%, amino acids selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R), providing the cells with fMLP and detecting phosphorylation of PKB (AKT) in the absence and presence of the peptide at an appropriate time interval, preferably in the order of minutes, most preferably from about half a minute to about 5 minutes, e.g., 30 to 600 seconds after provision of fMLP, and comparing the results to determine the peptide's effect on the phosphorylation.
  • the disclosure also provides a method for identifying a peptide capable of reducing PI3K/AKT/mTOR activity, comprising providing cells with a peptide consisting of amino acids, the amino acids for at least 50% selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R), providing the cells with fMLP and detecting phosphorylation of PKB (AKT) in the absence and presence of the peptide at an appropriate time interval, preferably in the order of minutes, most preferably from about half a minute to about 5 minutes, e.g., 30 to 600 seconds after provision of fMLP, and comparing the results to determine the peptide's effect on the phosphorylation.
  • alanine in one letter code: A
  • glutamine Q
  • G valine
  • V valine
  • L leucine
  • I isoleu
  • the disclosure therewith also provides method for identifying a peptide capable of reducing cytoskeleton reorganization, comprising providing cells with a peptide consisting of amino acids, the amino acids for at least 50% selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R), and providing the cells with fMLP and detecting phosphorylation of p38 MAPK and/or PKB (AKT) in the absence and presence of the peptide at an appropriate time interval, preferably in the order of minutes, most preferably from about half a minute to about 5 minutes after provision of fMLP, and comparing the results to determine the peptide's effect on the phosphorylation.
  • A alanine
  • Q glutamine
  • G glycine
  • V valine
  • L leucine
  • I proline
  • R arginine
  • MoA molecular mode-of-action
  • tissue-repair signals to the nutrient-sensing system of mTOR; leading to inhibition of autophagy and resulting in resolve of disease.
  • tissue-repair signal molecules change the balance of proteogenesis versus proteolysis in a cell of and lead to resolve of disease in three steps:
  • Administered peptide or amino acid fragments thereof are for taken up by amino acid transport, PEPT1/2 transport, by common endocytosis, in the case of vascular cells by elastin receptor-mediated endocytosis or phagocytosis.
  • amino acids inhibit autophagy, therewith inhibiting proteolysis and leading to proteogenic resolve and pharmaceutical effect.
  • the disclosure provides a tartrate or a citrate of a, preferably recombinant or synthetic, autophagy-inhibiting peptide, the peptide having an amino acid sequence comprising at least 50%, more preferably at least 75%, most preferably at 100% amino acids selected from the group of autophagy-inhibiting amino acids alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I) and proline (P).
  • A alanine
  • Q glutamine
  • G glycine
  • V valine
  • L leucine
  • I isoleucine
  • proline proline
  • the disclosure provides a stock solution, preferably aqueous, comprising a peptide-tartrate or a peptide citrate of a, preferably recombinant or synthetic, autophagy-inhibiting peptide, the peptide having an amino acid sequence comprising at least 50%, more preferably at least 75%, most preferably at 100% amino acids selected from the group of autophagy-inhibiting amino acids alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I) and proline (P).
  • A autophagy-inhibiting amino acids
  • A alanine
  • Q glutamine
  • G glycine
  • V valine
  • L leucine
  • I isoleucine
  • proline proline
  • the disclosure provides a suitable solution for several autophagy-inhibiting peptides to mitigate aggregation of the peptides and identifies tartrate (from tartaric acid, preferably from (+)-tartaric acid) and more preferably citrate (from citric acid) as a suitable counter-ion, pharmaceutical excipient or anion of choice for preparing a salt of an autophagy-inhibiting peptide that is a neutral peptide as defined herein above.
  • tartrate from tartaric acid, preferably from (+)-tartaric acid
  • citrate from citric acid
  • a variety of salts were screened herein to determine their influence on aggregation of neutral peptide according to the invention, indeed revealing that neutral peptide “salts out” of solution in an anion-specific and concentration-dependent manner.
  • a stock solution with an autophagy-inhibiting peptide invariably runs higher risks on peptide drug aggregation than a final working solution.
  • Stock solutions are generally prepared at a concentration well below an aggregation concentration of the salt in question (e.g., 40-50%) to prevent salt-out events under possibly prolonged storage at various ambient conditions.
  • Risk of peptide aggregation (salting-out) is a phenomenon that the disclosure provides to avoid or mitigate herein with a stock solution according to the invention.
  • Such stock solutions generally are diluted 10- to 100-fold, or more, to provide a suitable working solution. It is however also an object of the present disclosure to provide working solutions of the peptide-salts according to the invention. Particularly because in the application of the peptides of the disclosure relatively high amounts/concentrations of the peptide salts must be given, it is a prerequisite that the working solutions are far away from salting out points and yet are presented in a relatively small volume.
  • autophagy-inhibiting peptide herein is defined as a linear, branched or circular string of no longer than 50 amino acids that comprises a peptide sequence with at least 50%, more preferably at least 75%, most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), proline (P), isoleucine (I) and arginine (R).
  • MoA Molecular mode-of-action
  • the disclosure provides a peptide, preferably a salt of an organic acid, such as a maleate, more preferably an acetate, more preferably a tartrate, most preferably a citrate of a, preferably recombinant or synthetic, autophagy-inhibiting peptide, the peptide having an amino acid sequence comprising 50%, more preferably at least 75%, most preferably at 100% amino acids selected from the group of autophagy-inhibiting amino acids alanine (A), glutamine (Q), leucine (L), valine (V) glycine (G) and proline (P).
  • A alanine
  • Q glutamine
  • L leucine
  • V valine
  • G valine
  • P proline
  • the disclosure provides a stock solution, preferably aqueous, comprising a peptide-tartrate or a peptide citrate of a, preferably recombinant or synthetic, autophagy-inhibiting peptide, the peptide having an amino acid sequence comprising at least 50%, more preferably at least 75%, most preferably at 100% amino acids selected from the group of autophagy-inhibiting amino acids alanine (A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P).
  • A autophagy-inhibiting amino acids
  • A alanine
  • Q glutamine
  • G glycine
  • V valine
  • L leucine
  • P proline
  • a vial with a stock solution of AQGV (SEQ ID NO:2) peptide as defined here above for use in a clinical trial hitherto contained no more than (0.8 mol/L) active substrate in solution.
  • AQGV-salt (SEQ ID NO:2) of an organic acid in particular, of AQGV (SEQ ID NO:2) peptide-maleate, AQGV (SEQ ID NO:2) peptide-acetate AQGV (SEQ ID NO:2) peptide-tartrate or AQGV (SEQ ID NO:2) peptide-citrate (but not of adenosine or adenosine monophosphate) now is provided having an amino acid sequence comprising at least 50%, more preferably at least 75%, most preferably at 100% amino acids selected from the group of autophagy-inhibiting amino acids alanine (A), glutamine (Q), glycine (G), valine (V), leu
  • the disclosure provides a stock-solution of the AQGV (SEQ ID NO:2) peptide-tartrate or the AQGV (SEQ ID NO:2) peptide-citrate wherein the concentration of the AQGV (SEQ ID NO:2) peptide is in the range of 2 mol/L ( ) to 2.5 mol/L.
  • the disclosure provides a stock-solution of the AQGV-peptide-citrate wherein the concentration of the peptide-citrate is in the range of 2.5 mol/L to 3 mol/L.
  • the disclosure provides a stock-solution of the peptide-citrate wherein the concentration of the peptide-citrate is in the range of 3 mol/L to 3.5 mol/L. In a more preferred embodiment, the disclosure provides a stock-solution of the peptide-citrate wherein the concentration of the peptide-citrate is in the range of 3.5 mol/L to 4.5 mol/L. In a more preferred embodiment, the disclosure provides a stock-solution of the peptide-citrate wherein the concentration of the peptide-citrate is in the range of 4.5 mol/L to 5.5 mol/L. In a more preferred embodiment, the disclosure provides a stock-solution of the peptide-citrate wherein the concentration of the peptide citrate is equal to or larger than 5.5 mol/L. It is preferred that the stock solution is an aqueous solution.
  • the stock solution is an aqueous solution of autophagy-inhibiting amino acids comprising a dipeptide AQ, QQ, LQ, GQ, PQ, VQ, AL, LL, QL, GL, PL, VL, QA, QL, QG, QP, QV, LA, LG, LP, LV, a tripeptide AQG, QQG, LQG, GQG, PQG, VQG, ALG, LLG, QLG, GLG, PLG, VLG, QAG, QLG, QGG, QPG, QVG, LAG, LGG, LPG, LVG or a tetrapeptide AQGV (SEQ ID NO:2), QQGV (SEQ ID NO:24), LQGV (SEQ ID NO:25), GQGV (SEQ ID NO:26), PQGV (SEQ ID NO:27), VQGV (SEQ ID NO:28), ALGV (SEQ ID NO:29), LLGV (SEQ ID NO:
  • the disclosure provides a method for reducing p38 MAPK kinase activity leading to cytoskeleton reorganization, comprising providing cells, preferably having a formyl-peptide-receptor associated with their surface, with a source of autophagy-inhibiting amino acids, preferably wherein the source is an AQGV (SEQ ID NO:2) peptide as provided herein, the amino acids for least 50% selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R).
  • A alanine
  • Q glutamine
  • G glycine
  • V valine
  • L leucine
  • I proline
  • R arginine
  • the disclosure provides a method for reducing formyl-peptide-receptor (FPR)-mediated p38 MAPK kinase activity, comprising providing cells, preferably having a formyl-peptide-receptor associated with their surface, with a source of autophagy-inhibiting amino acids, preferably wherein the source is an AQGV (SEQ ID NO:2) peptide as provided herein, the amino acids for least 50% selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R).
  • FPR formyl-peptide-receptor
  • R arginine
  • the disclosure provides a method for reducing formyl-peptide-receptor (FPR)-mediated PI3K/AKT/mTOR activity, comprising providing cells, preferably having a formyl-peptide-receptor associated with their surface, with a source of autophagy-inhibiting amino acids, preferably wherein the source is an AQGV (SEQ ID NO:2) peptide as provided herein, the amino acids for least 50% selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R).
  • FPR formyl-peptide-receptor
  • the disclosure provides a method for reducing formyl-peptide-receptor (FPR)-mediated cytoskeleton reorganization, comprising providing cells, preferably having a formyl-peptide-receptor associated with their surface, with a source of autophagy-inhibiting amino acids, preferably wherein the source is an AQGV (SEQ ID NO:2) peptide as provided herein, the amino acids for least 50% selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R).
  • FPR formyl-peptide-receptor
  • the disclosure provides a method for modifying vascular permeability comprising providing cells, preferably having a formyl-peptide-receptor associated with their surface, with a source of autophagy-inhibiting amino acids, preferably wherein the source is an AQGV (SEQ ID NO:2) peptide as provided herein, selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R).
  • alanine in one letter code: A
  • glutamine Q
  • G glycine
  • V valine
  • L leucine
  • I isoleucine
  • P proline
  • R arginine
  • the disclosure provides a method for improving tissue repair comprising providing cells, preferably having a formyl-peptide-receptor associated with their surface, with a source of autophagy-inhibiting amino acids, preferably wherein the source is an AQGV (SEQ ID NO:2) peptide as provided herein, selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R).
  • alanine in one letter code: A
  • glutamine Q
  • G glycine
  • V valine
  • L leucine
  • I isoleucine
  • P proline
  • R arginine
  • the disclosure provides a method according to the invention, wherein the peptide comprising the autophagy-inhibiting amino acids comprises a dipeptide AQ, QQ, LQ, GQ, PQ, VQ, AL, LL, QL, GL, PL, VL, QA, QL, QG, QP, QV, LA, LG, LP, LV, a tripeptide AQG, QQG, LQG, GQG, PQG, VQG, ALG, LLG, QLG, GLG, PLG, VLG, QAG, QOLG, QGG, QPG, QVG, LAG, LGG, LPG, LVG or a tetrapeptide AQGV (SEQ ID NO:2), QQGV (SEQ ID NO:24), LQGV (SEQ ID NO:25), GQGV (SEQ ID NO:26), PQGV (SEQ ID NO:27), VQGV (SEQ ID NO:28), ALGV (SEQ ID NO:29),
  • this application finds the SARS-COV-2 spike protein (see also FIGS. 11 - 13 ) to carry a distinct peptide-motif sequence KWPWYIWL (SEQ ID NO:44) or variant KWPWYVWL (SEQ ID NO:45) in a membrane proximal external region (MPER) capable of binding to a receptor of the FPR-family of receptors.
  • COVID-19 is activating FPR-mediated pathways, in particular, through interaction of vascular cells with its spike protein, therewith leading to vascular leakage, thrombotic events and modulating Ang1/Ang2 ratio.
  • the MPER region may as well be involved in sparsely reported and incidental thrombotic events after vaccination with distinct coronavirus-based-vaccines that express the spike protein not fixated in a prefusion state.
  • AQGV SEQ ID NO:2
  • FPR-mediated pathways p38-MK2-HSP27 and PI3K-AKT-mTOR, see FIG.
  • AQGV-peptide improves vascular leakage and thrombotic events by inhibiting thrombus formation and modulating Ang1/Ang2 ratio after events causing expression of at least the fusogenic region with motif KWPWYIWL (SEQ ID NO:44) or variant KWPWYVWL (SEQ ID NO:45) in a subject.
  • the fusogenic region with motif KWPWYIWL (SEQ ID NO:44) or variant KWPWYVWL (SEQ ID NO:45) also comprises an FPR-binding site participating in inducing vascular leakage in a subject.
  • the present application also provides alternative treatment or use.
  • a method of treatment of a subject deemed to express a peptide or protein comprising a fusogenic region derived from a virus comprising adoptive cell therapy using at least one cell provided with a receptor recognizing the fusogenic site.
  • the fusogenic region at least comprises peptide motif KWPWYIWL (SEQ ID NO:44) or at least comprises peptide motif KWPWYVWL (SEQ ID NO:45), in particular, wherein the cell is a transformed T-cell, such as a CAR-T or TCR-T cell, preferably wherein the cell is directed against a (preferably CD8+) T-cell epitope comprising or overlapping the fusogenic region.
  • Such adoptive cellular therapy uses of at least one cell provided with a receptor recognizing a fusogenic region derived from a virus in method of treatment of a subject deemed to express a peptide or protein comprising the fusogenic region, wherein the fusogenic region at least comprises peptide motif KWPWYIWL (SEQ ID NO:44) or at least comprises peptide motif KWPWYVWL (SEQ ID NO:45), and wherein the cell is a transformed T-cell, such as a CAR-T or TCR-T cell preferably directed against a (preferably CD8+) T-cell epitope comprising or overlapping the fusogenic region.
  • a transformed T-cell such as a CAR-T or TCR-T cell preferably directed against a (preferably CD8+) T-cell epitope comprising or overlapping the fusogenic region.
  • An AQGV SEQ ID NO:2
  • SEQ ID NO:2 amino acid sequence of a human subject
  • a functional analogue thereof for use in the treatment of a human subject, the use comprising modifying hemodynamics in the human subject.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use in the treatment of a human subject, wherein the human subject is suffering from a viral infection and wherein the use comprises modifying hemodynamics in the human subject.
  • An AQGV SEQ ID NO:2
  • SEQ ID NO:2 amino acid sequence of a human subject having impaired lung function, the use comprising modifying hemodynamics and improving hypoxemia in the human subject.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use in accordance with any of further embodiments 1-4 wherein the use comprises a reduced use of vasopressive agents.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use in accordance with any one of further embodiments 1-5 wherein the use comprises a reduced fluid intake.
  • An AQGV SEQ ID NO:2 peptide, or a functional analogue thereof, for use in accordance with further embodiment 5, wherein the reduced use of vasopressive agents comprises a reduced duration of vasopressive agent use.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use in accordance with any one of further embodiments 3-6, wherein the subject is suffering from a respiratory viral infection.
  • An AQGV SEQ ID NO:2
  • SEQ ID NO:2 amino acid sequence of a human subject
  • a functional analogue thereof for use in accordance with any one of further embodiments 3-10, wherein the human subject has impaired lung function the impaired lung function being ARDS.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use as in accordance with any one of further embodiments 1-11, wherein the use reduces leakage of plasma from the blood to peripheral tissue and/or organs.
  • An AQGV SEQ ID NO:2 peptide, or a functional analogue thereof, for use in accordance with any one of further embodiments 1-12, wherein the use is in a human subject suffering from or at risk of suffering from detrimental effects of mechanical ventilation.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use in accordance with any one of further embodiments 1-13, wherein the use is in a human subject at risk of having edema.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use in accordance with any one of further embodiments 2-14, wherein the human subject is suffering or thought to be suffering from to corona virus infection.
  • An AQGV SEQ ID NO:2
  • SEQ ID NO:2 amino acid sequence of SEQ ID NO:2
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use in accordance with any one of further embodiments 1-20, wherein the human subject is admitted into intensive care, and wherein the use improves parameters measured of the human subject, the parameters of the human subject determined to assess remaining in intensive care.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use in accordance with further embodiment 21, wherein the improvement in parameters results in a reduced length of stay at intensive care.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use as in accordance with any one of further embodiments 1-22, wherein the uses induces vasoconstriction.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use as in accordance with any one of further embodiments 1-23, wherein the subject is deemed at risk of VALI or VILI.
  • An AQGV SEQ ID NO:2
  • peptide or a functional analogue thereof, for use as in accordance with any one of further embodiments 1-23, wherein the subject is deemed to express a peptide or protein comprising a fusogenic region derived from a virus.
  • a method of treatment comprising administering an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, to a human subject, the human subject being in need of maintaining hemodynamic stability.
  • a method of treatment comprising administering an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, to a human subject, the human subject being in need of improving hemodynamic stability.
  • a method of treatment comprising administering an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, to a human subject, the human subject having impaired lung function, wherein the treatment of administering an AQGV (SEQ ID NO:2) peptide comprises maintaining or improving hemodynamic stability in the human subject.
  • a method of treatment comprising administering an AQGV (SEQ ID NO:2) peptide, or a functional analogue thereof, intermittently to a human subject, the human subject having impaired lung function, wherein the treatment of administering an AQGV (SEQ ID NO:2) peptide comprises maintaining or improving hemodynamic stability in the human subject.
  • the fusogenic region at least comprises peptide motif KWPWYIWL (SEQ ID NO:44).
  • the fusogenic region at least comprises peptide motif KWPWYVWL (SEQ ID NO:45).
  • the cell is a transformed T-cell, such as a CAR-T or TCR-T cell.
  • FIG. 1 Early infections with the SARS-Cov-2 virus mostly run a mild or even uneventful course. That Stage I course is seen in >80% of people infected. This majority of patients experience an upper airway infection of nose and throat, with a dry cough, that generally passes in 2-12 days. In the remaining ⁇ 20% of cases, two distinct pathological stages may develop, often starting at around the time that in mild cases the viral infection is to reduce due to an emerging immune response directed against the virus.
  • Stage II is a viral pneumonia (pulmonary phase with lung injury) with permeability losses of the alveolar-capillary membrane (Stage IIA) that diffusely and profusely affects the deeper airways and alveoli of both lungs (Stage IIB), causing reduced uptake of oxygen due to respiratory failure. This two-sided pneumonia may be followed by stage III, a full-blown disease with general malaise, high fever and ultimately organ (kidney, liver, heart) failure at large.
  • ANG2 is stored in Weibel-Palade (WBP) bodies and rapidly released upon triggering signals. Its binding to TIE2 abolishes ANG1-induced sequestration of Src, resulting in the internalization of VE-cadherin.
  • WBP Weibel-Palade
  • angiopoietin-2 relative to angiopoietin-1 (Ang-2/Ang-1) may be a useful biologic marker of mortality in acute lung injury (ALI) patients.
  • FIG. 4 Schematic depiction in the infection stages of FIG. 1 of intermittent dosing of AQGV (SEQ ID NO:2)-peptide in various stages of COVID-19. Shadowed bars indicate time slots wherein AQGV (SEQ ID NO:2) peptide or related substance is administered intravenously, preferably at a rate of at least 75 mg/kg/hour, more preferably at least 90 mg/kg/hour. It is, in particular, useful to administer the AQGV (SEQ ID NO:2) peptide or related substance intermittently.
  • Preferred use is dosing for 2-4 hours of at least 75 mg/kg/hour, more preferably at least 90 mg/kg/hour, then optionally reducing to 30 mg/kg/hour for 2-4 hours (in between shaded bars), or for as long as it takes to monitor the patients response to treatment by clinical or laboratory diagnosis, or stop administering the substance for 1-2 hours until diagnostic studies have completed, and then resume treatment with 2-4 hours of at least 75 mg/kg/hour, more preferably at least 90 mg/kg/hour.
  • therapeutic effects of EA-230 may be monitored by determining hypoxia, plasma Ang2/Ang1 ratio and plasma levels of D-dimer in Stage II and III.
  • ICU intensive care unit
  • AQGV (SEQ ID NO:2)-peptide EA-230 reduced the number of patients at the ICU at 24 hours by 48%; and reduced hospital length of stay by 20%.
  • FIG. 7 Overview of solubility experiments with results in Table 1.
  • FIG. 8 Based on the results depicted in FIG. 7 the concentration below which an aggregated peptide-salt tends to resolve of the neutral-peptides salts screened were determined (aggregation points). It can be concluded that changing the anion significantly influences the solubility characteristics of AQGV (SEQ ID NO:2). Higher solubility (solubility in 0.9% NaCl) and therewith higher aggregation points were observed for the AQGV (SEQ ID NO:2)-citric acid (AQGV-citrate) and -tartaric acid (AQGV-tartrate (SEQ ID NO:2)) salt, whereas maleic acid and KHSO4 salts showed lower solubility, compared to AQGV-Ac (SEQ ID NO:2). Using adenosine-monophosphate or adenosine did not provide solubility. Citric acid seems to be a special case. Highly concentrated solution does not crystallize or aggregate but tend to form a highly viscous solution.
  • FIG. 9 Formyl-peptide-receptor-mediated vascular permeability after cell and tissue trauma.
  • the human formyl peptide receptor (FPR) is N-glycosylated and activates cells via G(i)-proteins. Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in cell membranes.
  • FPR human formyl peptide receptor
  • Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in cell membranes.
  • non-glycosylated FPR is much less active than native FPR.
  • Mitochondrial N-formyl peptides released from trauma/cell damage activate formyl peptide receptor (FPR) leading to changes in endothelial cell cytoskeleton, which subsequently induces endothelial contraction and vascular permeability, leukocyte extravasation and hypotension.
  • N-Formyl peptides are common molecular signatures of bacteria and mitochondria that activate the formyl peptide receptor (FPR).
  • FPR activation by mitochondrial N-formyl peptides (F-MIT) elicits changes in cytoskeleton-regulating proteins in endothelial cells that lead to increased endothelial cell contractility with increased vascular leakage and extravasation of leukocytes.
  • F-MIT mitochondrial N-formyl peptides
  • FIG. 10 Formyl-peptide-receptor-mediated peptide effects.
  • FPR-activation of FPR-expressing cells with prototype FPR-ligand fMLP causes rapidly induced and significant (p ⁇ 0.05; p38 from 60 to 600 sec, PKB at 600 sec) changes in phosphorylation status of PKB (also known as AKT) ( FIG. 10 a ) and p38 MAPK kinases ( FIG. 10 c ), but not (or not detected) in STAT3, JNK ( FIG. 10 b ) and P42/p44MAPK/ERK1,2 ( FIG. 10 d ) kinases.
  • AQGV peptide effects on p38 MAPK FIG.
  • AQGV (SEQ ID NO:2) peptide reduces p38 MAPK kinase activated changes as well as reduces PI3K/AKT/mTOR activated induced changes in cell cytoskeleton reorganization affecting endothelial cell contraction and adverse vascular permeability.
  • AQGV (SEQ ID NO:2) peptide is useful and capable of addressing adverse vascular permeability, such as manifested by edema with vascular leakage, adverse leukocyte extravasation and hypotension in human subjects.
  • FIG. 11 AQGV (SEQ ID NO:2)-peptide to target viral-spike-protein-induced pulmonary and vascular leakage in Coronavirus infections as seen in SARS, MERS and COVID-19.
  • SARS-CoV-2 spike (S) glycoproteins SEQ ID NO: 80-SEQ ID NO: 86
  • SEQ ID NO: 80-SEQ ID NO: 86 are class I viral fusion proteins that promote viral entry into cells and are the main target of antibodies (White et al., Critical reviews in biochemistry and molecular biology. 2008 Jan. 1; 43(3):189-219).
  • the C terminal end of spike protein contains a heptad repeat (HR2), a short linker region (the membrane proximal external region or MPER), a transmembrane helix domain (TMD) and a C-terminal cytoplasmic or internal domain (CTD/IC).
  • HR2 heptad repeat
  • MPER membrane proximal external region
  • TMD transmembrane helix domain
  • CCD/IC C-terminal cytoplasmic or internal domain
  • the heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains form a six-helix bundle fusion core (6HB), bringing the viral with the fusogenic MPER domain and cellular membranes together for fusion and cell entry (Walls et al., Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proceedings of the National Academy of Sciences. 2017 Oct. 17; 114(42):11157-62; Xia et al., Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cellular & molecular immunology. 2020 Feb. 11:1-3).
  • MPER is essential for viral entry into cells as identified in FIG. 12 .
  • at least one T cell epitope allowing generation of CD8+ T-cell cross-reactivity against SARS-CoV-2 and other coronavirus strains (Lee et al, Front. Immunol., 5 Nov. 2020 https://doi.org/10.3389/fimmu.2020.579480) overlaps with the fusogenic site as identified in FIG. 12 .
  • virus-specific CD4 + and CD8 + T cell responses are associated with milder disease, suggesting an involvement of the fusogenic region in protective immunity against COVID-19.
  • the fusogenic site, and therewith the T-cell epitope is strongly conserved in SARS-COV-2 (Guo E., Guo H. (2020) CD8 T cell epitope generation toward the continually mutating SARS-CoV-2 spike protein in genetically diverse human population: Implications for disease control and prevention.
  • PLOS ONE 15(12): e0239566) herein it is provided to develop adoptive cellular therapy (ACT) directed against the fusogenic region that may be used in viral or vaccine based infections such as with corona virus or vaccine.
  • ACT adoptive cellular therapy
  • FIG. 12 The short membrane proximal external region (MPER) connects the HR2 and transmembrane domain, and contains an aromatic-amino-acid-rich fusogenic peptide sequence that destabilizes the membrane during fusion (Mahajan M., Bhattacharjya S. NMR structures and localization of the potential fusion peptides and the pre-transmembrane region of SARS-CoV: Implications in membrane fusion. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2015 Feb. 1; 1848(2):721-30; Guéen J., Kinnunen P. K., Villalain J.
  • MPER The short membrane proximal external region
  • the MPER peptides 1185-LGKYEQYIKWPWYVWLGF-1202 (SEQ ID NO:87) and 1193-KWPWYVWLGFIAGLIAIV-1210 (SEQ ID NO:88) from SARS-CoV-1 have been shown to intercalate into lipid membranes and to be highly surface active; the corresponding fusogenic sequences in SARS-CoV-2 and MERS-CoV are identical except for a V to I substitution at position 1216 (SEQ ID NO:89-SEQ ID NO:91).
  • FIG. 13 Alignment of 36-mer AA sequences of near-C-terminal (position 1183-1218 in SARS-COV-2) with coronavirus spike proteins that have shown binding to FPR-receptor (with increased binding at polymorphism W190/N192). Synthesized 11/12-meric peptides having FPR-binding activity shown in red (SEQ ID NO:92-SEQ ID NO:97).
  • This application finds the SARS-COV-2 spike protein to carry a distinct and conserved fusogenic motif in its MPER domain (KWPWYIWL (SEQ ID NO:44)) that is capable of binding to FPR. This motif is highly homologous to related coronavirus spike protein motifs for which binding to FPR has been demonstrated.
  • Vascular leakage in COVID-19 is at least partly modulated by binding and/or fusing of this spike protein comprising at least the minimally essential fusogenic sequence KWPWYIWL (SEQ ID NO:44) or variant KWPWYVWL (SEQ ID NO:45) to pulmonary vascular cells carrying the formyl-peptide receptor, and therewith may cause thrombotic events in coronavirus infection or vaccination against corona with a spike protein-vaccine such as ChAdOx1-S, in particular, when such a vaccine is not modified to express the spike proteins in a prefusion state only.
  • a spike protein-vaccine such as ChAdOx1-S
  • FPR-mediated pathways are known to be activated in thrombotic events (Salamah et al., The formyl peptide fMLF primes platelet activation and augments thrombus formation. J. Thromb. Haemost. 2019; 17: 1120-1133) as well as in an acute lung injury and acute respiratory disease syndromes (ALI/ARDS) with pulmonary vascular leakage as a major clinical symptom (Thorax. 2017; 72:928-936).
  • COVID-19 in particular, the severe) infections with SARS-COV-2 typically increase risks on ALI/ARDS with pulmonary vascular leakage, leading to major fatalities.
  • amino acids In describing protein or peptide composition, structure and function herein, reference is made to amino acids. In the present specification, amino acid residues are expressed by using the following abbreviations. Also, unless explicitly otherwise indicated, the amino acid sequences of peptides and proteins are identified from N-terminal to C-terminal, left terminal to right terminal, the N-terminal being identified as a first residue.
  • Ala alanine residue; Asp: aspartate residue; Glu: glutamate residue; Phe: phenylalanine residue; Gly: glycine residue; His: histidine residue; Ile: isoleucine residue; Lys: lysine residue; Leu: leucine residue; Met: methionine residue; Asn: asparagine residue; Pro: proline residue; Gln: glutamine residue; Arg: arginine residue; Ser: serine residue; Thr: threonine residue; Val: valine residue; Trp: tryptophane residue; Tyr: tyrosine residue; Cys: cysteine residue.
  • “Peptide” shall mean herein a natural biological or artificially manufactured (synthetic) short chain of amino acid monomers linked by peptide (amide) bonds.
  • Glutamine peptide shall mean herein a natural biological or artificially manufactured (synthetic) short chain of amino acid monomers linked by peptide (amide) bonds wherein one of the amino acid monomers is a glutamine.
  • Chemically synthesized peptides generally have free N- and C-termini. N-terminal acetylation and C-terminal amidation reduce the overall charge of a peptide; therefore, its overall solubility might decrease. However, the stability of the peptide could also be increased because the terminal acetylation/amidation generates a closer mimic of the native protein.
  • peptides are either synthesized by classically known chemical synthesis on a solid support (Ansynth BV, Roosendaal, The Netherlands) or in solution (Syncom BV, Groningen, The Netherlands and Diosynth BV, Oss, The Netherlands).
  • Pharmaceutical peptide compositions may be synthesized using trifluoroacetate as a counter-ion or salt after which trifluoroacetate is exchanged by a counter-ion such as maleate (from maliec acid), acetate (from acetic acid), tartrate (from tartaric acid) or citrate (from citric acid).
  • EA-230 The drug substance of AQGV (EA-230) for use in pre-clinical and clinical human studies has been manufactured by Organon N.V (formerly Diosynth B.V.), (Oss, The Netherlands), whereas filling and finishing of the final product has been performed by Octoplus Development, Leiden (The Netherlands).
  • Molecular weight of EA-230 (AQGV) is 373 g/mol).
  • N-Formyl peptides are common molecular signatures of bacteria and mitochondria that activate the formyl peptide receptor (FPR).
  • FPR activation is a key contributor to impaired barrier function in following trauma. It has been proposed that in patients, mitochondrial components from damaged tissue can initiate the genesis of vascular leakage (Wenceslau et al., Front Immunol. 2016; 7: 297).
  • FPR formyl-peptide-receptor
  • the vasculature composed of vessels of different morphology and function, distributes blood to all tissues and maintains physiological tissue homeostasis.
  • the vasculature not only serves as the main carrier in gas exchange from lung to tissues (e.g., oxygen (and vice versa, e.g., carbon dioxide)) but also carries nutrients from gut to liver to tissues and toxic by-products resulting metabolism from tissues to kidney to urine for excretion.
  • the vasculature is often affected by, and engaged in, the disease process. This foremost results in adverse vascular permeability with edema, adverse vascular leakage, adverse leukocyte extravasation and hypotension and may also result in excessive formation of new, unstable, and hyper permeable vessels with poor blood flow, which further promotes hypoxia and disease propagation. Chronic adverse vessel permeability may also facilitate metastatic spread of cancer. Thus, there is a strong incentive to learn more about (and be able to modulate) an important aspect of vessel biology in health and disease: the regulation of vessel permeability.
  • Endothelial cells in different vessels and in different organs have distinct functions and morphologies (Aird W. C., Molecular heterogeneity of tumor endothelium. Cell Tissue Res. 2009; 335:271-81), but in general serve to provide a barrier between blood and tissue.
  • endothelial cells present certain morphological features that reflect the need for communication between the organs and the circulation.
  • the vasculature forms a particularly strong barrier, the blood-brain barrier (BBB) to protect the brain parenchyma from detrimental edema.
  • BBB blood-brain barrier
  • endothelial cells display specialized fenestrae on their surface. These are diaphragm-covered “holes” in the plasma membrane, which allow extremely rapid exocytosis of hormones.
  • the endothelial cells form a dynamic barrier between the blood and the tissue. In resting conditions, the vasculature continuously leaks solute and small molecules but restricts extravasation of larger molecules and cells. In many diseases, including cancer, the vascular barrier disintegrates and leakage increases and may become chronic. The leakage of larger molecules and cells may result in edema, adverse leukocyte extravasation and hypotension, and often disease progression.
  • kinins such as bradykinin are involved in a series of physiological and sometimes pathological vascular responses affecting endothelial barrier function. Most of their actions are mediated by the activation of 2 G protein-coupled receptors, named B1 and B2.
  • the activation of kinin receptors may play a key role in the modulation of atherosclerotic risk through the promotion of microangiogenesis, inhibition of vascular smooth muscle cell growth, coronary vasodilatation, increased local nitric oxide synthesis, or by exerting antithrombotic actions.
  • the bradykinin B1 receptor (B1R) is typically absent under physiological conditions, but is highly inducible following tissue injury, stress, burns, traumatic damage, such as, for example, recently reported in COVID-19 disease.
  • Damage induced by tissue injury may cause a significant and time-dependent increase in des-Arg9-bradykinin (des-Arg9-BK) responsiveness that parallels B1R mRNA expression. It induces the activation of some members of the mitogen activated protein kinase (MAPK) family, namely, extracellular signal-regulated kinase (ERK) and p38 MAPK.
  • MAPK mitogen activated protein kinase
  • ERK extracellular signal-regulated kinase
  • p38 MAPK extracellular signal-regulated kinase
  • the blockade of p38 MAPK but not ERK pathways with selective inhibitors results in a significant reduction of the upregulated contractile response caused by the selective B1R agonist des-Arg9-BK, and largely prevents the induction of B1R mRNA expression enhancing tissue damage induced adverse vascular permeability.
  • cytoskeletal changes in response to stress involves the reorganization of the actin cytoskeleton and the formation of stress fibers.
  • Kayyali et al. J. Biol. Chem. (2002) 277(45): 42596-602 describe cytoskeletal changes in pulmonary microvascular endothelial cells in response to hypoxia and potential mechanisms involved in this process.
  • the hypoxia-induced actin redistribution appears to be mediated by components downstream of MAPK p38, which is activated in pulmonary endothelial cells in response to hypoxia.
  • Results indicate that kinase MK2, which is a substrate of p38, becomes activated by hypoxia, leading to the phosphorylation of one of its substrates, HSP27.
  • HSP27 a target of p38 MAPK/MK2 pathway
  • HSP27 phosphorylation is known to alter actin distribution and thus contractility of cells
  • Kayyali et al. provide that the p38-MK2-HSP27 pathway causes changes in vascular permeability due to actin redistribution, as, for example, observed in hypoxia.
  • PI3K/AKT/mTOR phosphatidylinositol 3′ kinase (PI3K), protein kinase B (PKB or AKT) and mammalian target of rapamycin (mTOR)] pathway
  • PI3K phosphatidylinositol 3′ kinase
  • PKT protein kinase B
  • mTOR mammalian target of rapamycin
  • Such elongation is required to fill the gaps that form between endothelial cells when these cells contract after p38-MK2-HSP27 and/or PI3K/AKT/mTOR signaled cytoskeleton reorganization. It is these gaps (again see FIG. 1 ) through which adverse leakage and adverse extravasation occurs that explains the resulting edema, vascular leakage, adverse leukocyte extravasation and loss of vascular fluid with a risk for hypotension.
  • Closing of these gaps is in general governed by the ratio of various angiogenic factors such as angiopoietin-2 to angiopoietin-1 at the site of increased vascular permeability, whereby angiopoetin-2 in general induces endothelial cell apoptosis (there with enhancing gap-formation) and angiopoietin-1 counters gap formation by facilitating endothelial vcel elongation and gap closure. Inhibition of the p38 pathway, but not of the ERK1/2 pathway, attenuates angiopoetin-2-mediated endothelial cell apoptosis (Li et al., Exp. Ther. Med.
  • PI3K/AKT/mTOR pathway modulates the expression of other angiogenic factors as nitric oxide and angiopoietins (Karar and Mayti, Front. Mol. Neurosci., 2 Dec. 2011, https://doi.org/10.3389/fnmo1.2011.00051).
  • FIGS. 1 , 2 and 3 In the remaining cases distinct pathological states may develop ( FIGS. 1 , 2 and 3 ), often starting at around the time that in mild cases the viral infection is considered bound to reduce due to an emerging immune response directed against the virus.
  • This two-sided pneumonia may be rapidly followed by full-blown systemic disease with general malaise, high fever and ultimately organ (kidney, liver, heart) failure.
  • ICU intensive care unit
  • AQGV SEQ ID NO:2
  • VALI Ventilator-Associated-Lung-Injury
  • VILI Ventilator-Induced-Lung-Injury
  • VALI and VILI are recognized herein to play a distinct role in accelerating multiple organ failure associated with COVID-19.
  • Patients requiring mechanical ventilation consume a disproportionately high amount of healthcare resources, both in the ICU and after hospital discharge. Their short-term and long-term mortality is high, and they suffer a very heavy symptom burden for prolonged periods. Hospital survivors have a significant degree of functional and cognitive limitations, and a high readmission rate. Some remain at high risk for death after hospital discharge. Prolonged hospitalization for PMV patients who are at high risk of death does not meet current standards of cost-effectiveness.
  • AQGV Peptide EA-230 Reduces Adverse Vascular Fluid Permeability.
  • EA-230 has surprisingly been found to modulate vascular permeability to the good.
  • EA-230 has a very short half-life, which facilitates intermittent dosing of the drug and determination of its actual effects at bedside to determine progress of the patient during treatment and make rapid decisions about continuation or discontinuation of treatment.
  • EA230 exhibited a very short elimination half-life and a large volume of distribution (LPS-study: geometric mean and 95% confidence interval: 0.17 [0.12-0.24] hours and 2.2 [1.3-3.8] L/kg, respectively).
  • Respiratory failure is a common complication not only of COVID-19 and flu but of other respiratory diseases caused by coronaviruses such as SARS and MERS.
  • SARS and MERS coronaviruses
  • the phenomenon became more widely known after the 2005 outbreak of the avian H5N1 influenza virus, also known as “bird flu,” when the high fatality rate was linked to an out-of-control systemic multi-organ failure.
  • bird flu avian H5N1 influenza virus
  • Vaccines and antivirals may differ, fighting respiratory failure stays the same.
  • AQGV peptide can still be used.
  • EA-230 Early administration led us detect novel and truly beneficial effects of EA-230 on hemodynamics, kidney function, length of stay in ICU and hospital, that relate to improved hemodynamic stability.
  • Treatment of patients with EA-230 during surgery significantly reduced the need for hemodynamic therapy (combined fluid therapy and blood pressure medication; p 0.006).
  • a prospective, randomized, double-blind, placebo-controlled study was performed in which 180 elective patients undergoing on-pump coronary artery bypass grafting were enrolled. Patients were randomized in a 1:1 ratio and received either EA-230, 90 mg/kg/hour, or a placebo. These were infused at the start of the surgical procedure until the end of the use of the cardiopulmonary bypass.
  • the main focus in this first-in-patient study was on safety and tolerability of EA-230.
  • the primary efficacy endpoint was the modulation of the inflammatory response by EA-230.
  • a key secondary endpoint was the effect of EA-230 on renal function.
  • the present study was a single-center, prospective, double-blind, placebo-controlled, randomized, single-dose phase II study. It has an adaptive design to evaluate the safety and immunomodulatory effects of EA-230 in patients undergoing for coronary artery bypass grafting (CABG). 180 eligible patients were included and were randomized to receive either active or placebo treatment in a 1:1 ratio. This was a first-in-patient safety and tolerability study, of which the primary efficacy objective was to assess the immunomodulatory effects of EA-230. The key-secondary efficacy endpoint was the effect of EA-230 on renal function. This study was described in accordance with the Standard Protocol Items: Recommendations for Interventional Trial (SPIRIT) guidelines, and registered at clinicaltrials.gov under number NCT03145220.
  • Double-blind conditions were maintained for all patients, the attending physicians and the medical study team personnel involved in all blinded study procedures, data collection and/or data analyses.
  • Non-blinded study personnel not involved in any other study procedures prepared the study medication.
  • Infusion systems and solutions for active and placebo treatment were identical in appearance and texture. Unblinding was authorized by the sponsor after completion of the study, performance of a blinded data review and locking of the database.
  • Intravenous infusion of EA-230, 90 mg/kg/hour, or placebo was initiated at the moment of first surgical incision using an automated infusion pump. Infusion rate was set at 250 mL/hour, and infusion was continued until cessation of the CPB, or after 4 hours of continuous infusion, whichever comes first.
  • the EA-230 formulation was packed in sterile 5 mL glass vials, containing 1500 mg/vial, dissolved in water for injection at a final concentration of 300 mg/mL with an osmolality of 800 to 1000 mOsm/kg.
  • the placebo formulation consisted of sodium chloride diluted in water for injection in identical sterile 5 mL glass vials containing 29 mg/mL to reach a solution with an identical osmolality.
  • EA-230 and placebo were prepared for continuous intravenous infusion with an osmolality of ⁇ 400 mOsm/Kg by adding the appropriate amount of EA-230 or placebo to 1000 mL normal saline under aseptic conditions.
  • Placebo and active treatment vials were manufactured by HALIX BV (Leiden, the Netherlands).
  • SAEs or SUSARs include death, life-threatening disease, persistent and/or significant disability and/or incapacity, and hospitalization and/or prolongation of inpatient hospitalization.
  • AQGV SEQ ID NO:2
  • vasopressors were reduced in the group that was treated with EA-230. Patients were divided in quartiles based on treatment duration. In Table 3, descriptive frequencies of the 2 variables: days on vasopression and nett fluid balance day 0-2 (first 72 hours) are shown. The groups were split in patients without acute kidney injury (AKI) and with AKI, as well in patients without treatment (placebo) and with treatment with EA-230 (active). EA-230 decreased the net (netto) fluid balance in patients both with and without AKI. EA-230 decreased the need for vasopressors in patients with AKI.
  • AKI acute kidney injury
  • Placebo patients without treatment
  • EA-230 active
  • EA-230 decreased the net (netto) fluid balance in patients both with and without AKI.
  • EA-230 decreased the need for vasopressors in patients with AKI.
  • Input/independent variable treatment group (EA-230 or placebo).
  • Output/dependent variables were: endpoint of fluid balance first 72 hours, days on vasopressin or vasopressor score (area under the curve).
  • Effects of EA-230 versus placebo were tested on two combined variables in model A: fluid balance first 72 hours+days on vasopressin and model B: Fluid balance first 72 hours+vasopressor score AUC.
  • model A fluid balance first 72 hours+days on vasopressin
  • model B Fluid balance first 72 hours+vasopressor score AUC.
  • EA-230 SEQ ID NO:2
  • EA-230 (SEQ ID NO:2) has an Advantageous Effect on Kidney Function
  • EA-230 Effects of EA-230 on modulation in incidence of different stages of acute kidney injury (AKI) were determined according to the RIFLE criteria (RIFLE: risk, injury, failure, loss of kidney function, and end-stage kidney disease classification, Clin. Kidney J. 2013 February; 6(1): 8-14).
  • RIFLE risk, injury, failure, loss of kidney function, and end-stage kidney disease classification
  • the number of patients with no AKI increased, whereas the number of patients in the Injury category of the RIFLE criteria decreased.
  • GFR Creatinine clearance, a biomarker of kidney function, was significantly improved in patients treated with EA-230. When kidney function was taken into account, clearance of creatinine was significantly improved when EA-230 was used, when kidney function was below 60 mL/minute.
  • kidney function was above 60 mL/minute, no differences were observed.
  • pre-treatment kidney function was above 60 mL/minute/1.73 m2, no differences were found between groups.
  • CABG-study The number of patients that had to be re-admitted to the hospital due to clinical disease in the period post-treatment. Readmittance was scored in the period of 28 days after operation, and in the period ranging from 29-90 days after operation, and for the total period of 90 days after operation. Readmittance was reduced in patients receiving EA-230 treated group.
  • EA-230 Treatment with EA-230 herewith shows strong beneficial effects on recovery.
  • EA-230-treated patients required significantly less hemodynamic therapy, regained post-surgical kidney function significantly faster and remained for a shorter period of time in the Intensive Care Unit (ICU) and in the hospital, as compared to placebo-treated patients.
  • ICU Intensive Care Unit
  • EA-230 novel hemodynamic effects of EA-230 are independent of anti-inflammatory effects of EA-230.
  • significant improvements of hemodynamic stability, kidney function and recovery of EA-230 treated patients relate to novel effects of EA-230 on blood vessel-permeability and blood vessel-contractility.
  • EA-230 shows significant improvements in patient recovery, over placebo patients.
  • IL-6 long-term inflammatory cytokine
  • Plasma samples are further analyzed with regard to selected biomarkers.
  • Plasma samples of control patients and patients receiving the EA-230 are analyzed with regard to biomarkers Endothelin-1, VEGF, Angiotensin II, ANG2/ANG1 ratio, and cAMP and natriuretic peptides.
  • AQGV peptide EA-230
  • analogues thereof is tested on human endothelial cells.
  • endothelial cells are cultured in transwell culture dishes and culture medium is supplemented with AQGV (SEQ ID NO:2) peptides, and analogues thereof, or control compounds known to affect endothelial layer permeability, vasoconstriction and/or vasodilation.
  • AQGV SEQ ID NO:2
  • Suitable human endothelial cells are, e.g., HUVECs (Park et al., Stem Cell Rev.
  • HMEC-1 Ades E W, et al., J. Invest. Dermatol. 99(6): 683-690, 1992.
  • the permeability of the endothelial layer is determined by measuring the penetration of a macromolecule. Furthermore, levels of biomarkers are also determined in culture medium. Experiments are carried as outlined, e.g., in Cox et al., Shock, 43(4):322-6; 2015.
  • HUVEC human endothelial vascular cells
  • HaSMC human aortic smooth muscle cells
  • effectors such as thrombin, bradykinin, lipopolysaccharide, (LPS), spike protein of coronavirus, nucleic acid of coronavirus, high mobility group box
  • EA-230-peptide or placebo controls Similar studies are used to various test-concentrations of EA-230-peptide or placebo controls used, detecting effects of EA-230 in human lung organoid cultures, with or without effectors, such as thrombin, bradykinin, lipopolysaccharide, (LPS), spike protein of coronavirus, nucleic acid of coronavirus, high mobility group box 1 (HMGB1) protein, and reversing their effects with AQGV-peptides.
  • effectors such as thrombin, bradykinin, lipopolysaccharide, (LPS), spike protein of coronavirus, nucleic acid of coronavirus, high mobility group box 1 (HMGB1) protein, and reversing their effects with AQGV-peptides.
  • EA-230-peptide or placebo controls Similar studies are used to various test-concentrations of EA-230-peptide or placebo controls used, detecting effects of EA-230 in experimental mice provided with human ACE2 receptor, with or without effectors, such as thrombin, bradykinin, lipopolysaccharide, (LPS), spike protein of coronavirus, nucleic acid of coronavirus, high mobility group box 1 (HMGB1) protein, and reversing their effects with AQGV-peptides.
  • effectors such as thrombin, bradykinin, lipopolysaccharide, (LPS), spike protein of coronavirus, nucleic acid of coronavirus, high mobility group box 1 (HMGB1) protein, and reversing their effects with AQGV-peptides.
  • compositions for use in method of reducing the permeability of an endothelial layer of a blood vessel in a subject comprising:

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